Transcriptomic and Metabolomic Profiling Provides Insights into Flavonoid Biosynthesis and Flower Coloring in Loropetalum chinense and Loropetalum chinense var. rubrum

Round 1

Reviewer 1 Report

Zhang et al, submitted manuscript, "Transcriptomic and metabolomic profiling provides insights into flavonoids biosynthesis and flower coloring in Loropetalum chinense and Loropetalum chinense var. rubrum" details an interesting study related to the flower-derived coloring pigments (anthocyanins).

There are some minor comments.

1. Some suggestions on the choice of words.

Line 29, "The total anthocyanins and phenotypic of the petal..." check "phenotypic" if it is correct?

Line 30 and 31, "Of these, 12 flavonoid components were considered significantly different expression compounds among the four samples"

This line needs some attention.

Also, sometimes authors wrote compounds and, other times, components; please make it more uniform.

2. For Identifying the petals of flowering of particular species, respective morphological and anatomical characteristics must be provided in the material and method section, so that other researchers can proficiently replicate the current study.

3. The methodology of estimating the specific anthocyanin is unclear. Comparing with previous studies was the only way the author estimated the anthocyanins in samples? Please clarify.

The English of the paper requires some attention.

Author Response

Dear Reviewer,

 

Thank you for your detailed review of our manuscript entitled “Transcriptomic and metabolomic profiling provides insights into flavonoids biosynthesis and flower coloring in Loropetalum chinense and Loropetalum chinense var. rubrum” (2252556). The comments are of great help to improving the manuscript. We have studied the comments carefully and perform corresponding corrections in the revised manuscript. The point-by-point responses to the comments and suggestions are listed below.

 

Zhang et al, submitted manuscript, "Transcriptomic and metabolomic profiling provides insights into flavonoids biosynthesis and flower coloring in Loropetalum chinense and Loropetalum chinense var. rubrum" details an interesting study related to the flower-derived coloring pigments (anthocyanins). There are some minor comments.

(1)  Some suggestions on the choice of words.

Line 29, "The total anthocyanins and phenotypic of the petal..." check "phenotypic" if it is correct?

Line 30 and 31, "Of these, 12 flavonoid components were considered significantly different expression compounds among the four samples"

This line needs some attention.

Also, sometimes authors wrote compounds and, other times, components; please make it more uniform.

(2) For Identifying the petals of flowering of particular species, respective morphological and anatomical characteristics must be provided in the material and method section, so that other researchers can proficiently replicate the current study.

(3). The methodology of estimating the specific anthocyanin is unclear. Comparing with previous studies was the only way the author estimated the anthocyanins in samples? Please clarify.

 

 

   Q1: Some suggestions on the choice of words. Line 29, "The total anthocyanins and phenotypic of the petal..." check "phenotypic" if it is correct? Line 30 and 31, "Of these, 12 flavonoid components were considered significantly different expression compounds among the four samples" This line needs some attention. Also, sometimes authors wrote compounds and, other times, components; please make it more uniform.

Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we reversed our abstract as below: (Line 29 – Line 30) “The total anthocyanins, and RHSCC values and CIE 1976 L*a*b* values of the petal were highly consistent with the petal color. ” ; And (Line 31 – Line 32) “ Of these, 12 flavonoid compounds were considered significantly different expression compounds among the four samples.”. Meanwhile, we have checked all the “compounds” and “components” in our manuscript where it is should be.

 

Q2: For Identifying the petals of flowering of particular species, respective morphological and anatomical characteristics must be provided in the material and method section, so that other researchers can proficiently replicate the current study.

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have added the anatomical characteristics method and results. The method in Line 141 – Line 146: “2.3 Transverse section observation. For transverse section observations, the different Loropetalum spp. petals were cut into thin slices by using razor blade, respectively. And then the thin slice was fully ex-tended flatting on temporary slide with a clean filter covering. Next, the temporary glazing of petals was observed and taken photos with microscope (DMIL LED, Leica, Wetzlar, Germany) and its imaging system (LEICA DFC295 and Leica Microsystems CMS GmbH, Leica, Wetzlar, Germany).” The results in Line 293 – Line 295: “The transverse section of petals was shown that the distribution of anthocyanins was significant different in four Loropetalum spp.(Figure 1B).”; and the Figure 1 as below:

Figure 1.Phenotypes of Loropetalum cultivars and total anthocyanin content. (A). Phenotypes of four Loropetalum cultivars, namely, 'Xiangnong Xiangyun' (XNXY), 'Huye Jimu 2' (LDPF), 'Xiangnong Fenjiao' (XNFJ) and 'Xiangnong Nichang' (XNNC) of 2 days after the flowering stage, scalebars=0.5 cm. (B). Transverse section of petals of four Loropetalum cultivars, namely, 'Xiangnong Xiangyun' (XNXY), 'Huye Jimu 2' (LDPF), 'Xiangnong Fenjiao' (XNFJ) and 'Xiangnong Nichang' (XNNC), scalebars=100 μm. (C). Total anthocyanin content in the petals of four Loropetalum cultivars, namely, 'Xiangnong Xiangyun' (XNXY), 'Huye Jimu 2' (LDPF), 'Xiangnong Fenjiao' (XNFJ) and 'Xiangnong Nichang' (XNNC). Error bars indicate standard error (+SE) of the mean. Different letters indicated significant differences at p≤0.05 level basing on Duncan's test.

 

Q3: The methodology of estimating the specific anthocyanin is unclear. Comparing with previous studies was the only way the author estimated the anthocyanins in samples? Please clarify.

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we redescribed the protocol of methodology of estimating the specific anthocyanin. we reversed our manuscript as below: (Line 186 – Line 193) “The Mass Bank, KNAPSACK, HMDB, MOTDB and METLIN were used to analyze the structure of metabolites. The KEGG pathway public database was used to identified the specific metabolic pathways. The qualitative and quantitative of each flavonoid metabolites was identified and quantified based on the MWDB and open public metabolite database. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period. The target substance was screened and identified by parent ion (Q1), fragment ions (Q2) and the characteristic fragment ions (Q3) [6,37].”

 

Once again, thank you very much for your comments and suggestions. A revised manuscript is attached. Should you have any questions, please contact us without any hesitation.

Sincerely yours,

Corresponding author

Yanlin Li

27^th April, 2023

Author Response File: Author Response.pdf

Reviewer 2 Report

In this study, Zhang et al. conducted transcriptome and metabolome analyses for the petal color formation in Loropetalum chinense and L. chinense var. rubrum. Authors wrote the manuscript very nicely. However, there are still some problems that need to be communicated.

 

1. Cyanidin 3-O-glucoside was the major compound in three colored petals, which needs to be more described and discussed.

2. Transcriptional regulation is important for anthocyanin biosynthesis, such as MYB and bHLH. The results of TFs identification, expression and correlation analysis should be added.

3. All the Latin name should be italic (like, L191, 291, 622 et al.). And also for the L, a, b index.

4. L145-147, unclear.

5. L259, DETs with fold change ≥ 2 represented the up-regulated ones. How about the down-regulated transcript (fold change ≤ 0.5)?

6. L277, add the color of LDPF.

7. L305, List the top 5/10 metabolites for each sample. And more, the correlations between Lab index and main metabolites or class contents could suggest the color formation more clearly.

8. L364-365, How many DAMs for each pathway?

9. L442-444, unclear. The up- and down-regulated DEGs for 5 compare combinations?

10. L537, The figure of correlation analysis could be presented in the manuscript. structural gene—metabolites, TFs—metabolites, structural gene—TFs.

11. L558-561, List the index in the Result section (Same as No. 3)

12. 583-600, This part is redundant, just one or two sentences will be Okay. The authors should discuss the difference of metabolites, structural genes and TFs most, and then identify the key genes and TFs related to color modification in the petals of Loropetalum chinense and L. chinense var. rubrum.

13. The Methods section is too detailed and should be condensed.

14. The authors listed 34 tables and 18 figures in the supplementary materials. Some figures and tables should be combined, like Fig. -S14, Table S2-S7, S9-S14, S15-S20…

15. The order of four cultivars should be consistent.

16. There are two colors in the petals of LDPF. The authors should explain the process of sample collection.

See above, please.

Author Response

Dear Reviewer,

 

Thank you for your detailed review of our manuscript entitled “Transcriptomic and metabolomic profiling provides insights into flavonoids biosynthesis and flower coloring in Loropetalum chinense and Loropetalum chinense var. rubrum” (2252556). The comments are of great help to improving the manuscript. We have studied the comments carefully and perform corresponding corrections in the revised manuscript. The point-by-point responses to the comments and suggestions are listed below.

 

In this study, Zhang et al. conducted transcriptome and metabolome analyses for the petal color formation in Loropetalum chinense and L. chinense var. rubrum. Authors wrote the manuscript very nicely. However, there are still some problems that need to be communicated.

(1) Cyanidin 3-O-glucoside was the major compound in three colored petals, which needs to be more described and discussed.

(2) Transcriptional regulation is important for anthocyanin biosynthesis, such as MYB and bHLH. The results of TFs identification, expression and correlation analysis should be added.

(3) All the Latin name should be italic (like, L191, 291, 622 et al.). And also for the L, a, b index.

(4) L145-147, unclear.

(5) L259, DETs with fold change ≥ 2 represented the up-regulated ones. How about the down-regulated transcript (fold change ≤ 0.5)?

(6) L277, add the color of LDPF.

(7) L305, List the top 5/10 metabolites for each sample. And more, the correlations between Lab index and main metabolites or class contents could suggest the color formation more clearly.

(8) L364-365, How many DAMs for each pathway?

 

(9) L442-444, unclear. The up- and down-regulated DEGs for 5 compare combinations?

(10) L537, The figure of correlation analysis could be presented in the manuscript. structural gene—metabolites, TFs—metabolites, structural gene—TFs.

(11) L558-561, List the index in the Result section (Same as No. 3)

(12) 583-600, This part is redundant, just one or two sentences will be Okay. The authors should discuss the difference of metabolites, structural genes and TFs most, and then identify the key genes and TFs related to color modification in the petals of Loropetalum chinense and L. chinense var. rubrum.

(13) The Methods section is too detailed and should be condensed.

(14) The authors listed 34 tables and 18 figures in the supplementary materials. Some figures and tables should be combined, like Fig. -S14, Table S2-S7, S9-S14, S15-S20…

(15) The order of four cultivars should be consistent.

(16) There are two colors in the petals of LDPF. The authors should explain the process of sample collection.

 

   Q1: Cyanidin 3-O-glucoside was the major compound in three colored petals, which needs to be more described and discussed.

Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we reversed our manuscript as below: (Line 360 - Line 368) “three Anthocyanidin (Peonidin, Petunidin 3-O-glucoside, and Peonidin 3-sophoroside-5-glucoside), one Flavanone (Hesperetin O-Glucuronic acid), six Flavone (Chrysoeriol 6-C-hexoside, di-C,C-hexosyl-apigenin, 8-C-hexosyl chrysoeriol O-hexoside, Tricin 7-O-hexosyl-O-hexoside, Acacetin O-glucuronic acid and Apigenin 6,8-C-diglucoside), and two flavonol (Kaempferide, Ayanin) were found in HJ, XN, and XF, respectively. Moreover, one flavone (C-hexosyl-luteolin O-hexoside) and one Isofla-vone (Glycitin) were detected in XX (Figure 2C, Supplementary table S8). In addition, Cyanidin 3-O-glucoside was found in XF, XN, XX, and HJ, significantly different in the four samples(Figure 2C, Supplementary table S8)”.

 

Q2: Transcriptional regulation is important for anthocyanin biosynthesis, such as MYB and bHLH. The results of TFs identification, expression and correlation analysis should be added.

      Response: Thanks for the above suggestion. We agree that transcriptional regulation is vital for anthocyanins biosynthesis, such as MYB, bHLH, and WD40.  Indeed, we analyzed the transcription factor characteristic and identified the transcription factor in our manuscript in Line 444 – Line 449 : “In total, 5487 TFs were identified and divided into more than 28 families (Figure S7B). The most abundant family was C2H2 (397 of all), followed by C3H (341 of all), bHLH (324 of all), FAR1 (291 of all), and SNF2 (254 of all). In addition, 26,590 LncRNAs were predicted from the final transcripts, ranging from 200 to 11,293 bp (Figure S7C, Figure S7D). These data provided an abundant gene and transcript pool for further study of metabolic process.”. While readcounts for each transcript was estimated by the expected number of Fragments Per Kilobase of transcript sequence per Millions of base pairs sequenced (FPKM)  method by using RESM. And then, Genes and transcripts with fold change ≥2 and adjusted p-value ≤0.05 were considered significant DEGs and DETs. The gene ontology (GO) enrichment of all the DEGs and DETs was performed using the GO-seq R packages. KEGG database and KOBAS software were subjected to KEGG Pathway enrichment analysis, and significantly enriched metabolic or signal transduction pathways select with an adjusted p value of ≤0.05. Combined with the transcriptome and metabolome results, we just got the main functional genes of flavonoid and anthocyanins biosynthesis, a significant differential expressed in the four samples. So, we didn’t add the results of TFs identification, expression and correlation analysis.

 

Q3: All the Latin name should be italic (like, L191, 291, 622 et al.). And also for the L, a, b index.

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have changed all the Latin name and L, a and b index as italic in all of our manuscript.

 

Q4: L145-147, unclear.

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have changed it to “And then transferred 1 ml supernatant into a clean tube with 4 mL of buffer A (0.4 M KCl, pH 1.0) and buffer B (1.2 N citric acid, pH 4.5), respectively. The mixture took absorbance measurements at 510 and 700 nm for A and B mixtures separately.” in Line152 -Line155.

 

Q5: L259, DETs with fold change ≥ 2 represented the up-regulated ones. How about the down-regulated transcript (fold change ≤ 0.5)?

      Response: Thanks for the above suggestion. We apologize for the misunderstanding of the description on the DEGs screening. We described “Genes and transcripts with fold change ≥2 and adjusted p-value ≤0.05 were considered significant DEGs and DETs.” ln Line 259. We didn’t indicate that DETs with fold change ≥ 2 represented the up-regulated ones, and fold change ≤ 0.5 represented the down-regulated transcript. DESeq2 software was used to identify the differentially expressed genes (DEGs), and just defined that the selected among all genes with a filter of |log2 fold change| ≥ 2 and adjusted p-value ≤0.05 were considered significant DEGs and DETs. So, based on your suggestion, we have changed “Genes and transcripts with fold change ≥2 and adjusted p-value ≤0.05 were considered significant DEGs and DETs.” to “Genes and transcripts with |log2 fold change| ≥2 and adjusted p-value ≤0.05 were considered significant DEGs and DETs.” in Line 272 to Line 273.

 

Q6: L277, add the color of LDPF.

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have added the color of HJ(LDPF) as below: “(White part: Royal Garden Color Card, NN155C; purple part: Royal Garden Color Card, 61A)” in Line 290 - Line 291.

 

Q7: L305, List the top 5/10 metabolites for each sample. And more, the correlations between Lab index and main metabolites or class contents could suggest the color formation more clearly.

      Response: Thanks for the above suggestion. We have listed all the metabolites for each sample in supplementary Table S1, which include the top 5/10 metabolites. We analyze the main metabolites, including class contents with the sample’s color formation. The compared results of each different color petal described as L*, a*, and b* value index can be found in Figure 2, Figure S2 (the top 20 metabolites between each group of two different color petals),  and supplementary Table S9 to supplementary Table S20. In addition, the protocol of qualitative and qualification of flavonoid is semiquantitative by using a widely targeted metabolome method (Han Li, Zhen Yang, Qiwei Zeng, Shibo Wang, Yiwei Luo, Yan Huang, Youchao Xin, Ningjia He, Abnormal expression of bHLH3 disrupts a flavonoid homeostasis network, causing differences in pigment composition among mulberry fruits, Horticulture Research, Volume 7, 2020, 83, https://doi.org/10.1038/s41438-020-0302-8; Li, Y., Chen, Y., Zhou, L., You, S., Deng, H., Chen, Y., ... & Zhang, Y. (2020). MicroTom metabolic network: rewiring tomato metabolic regulatory network throughout the growth cycle. Molecular plant, 13(8), 1203-1218.), each metabolite content in one sample has litter biological significance if they haven’t compared with a control.

 

Q8: L364-365, How many DAMs for each pathway?

      Response: Thanks for the above suggestion. We have shown the results in supplementary Table S15 to Table S20. And we showed the DAMs results by combining the table below (Table 1). And the metabolites’ names of each pathway can be found in supplementary Table S15 to Table S20 in Colum of Index.

Table 1 The number of DAMs for each pathway

The compared transcriptome of HJ VS XN on KEGG metabolic pathway annotation
Pathway	ko_ID	Unique compound	compound	Uni_all	compound_all
Flavonoid biosynthesis	ko00941	15	36	26	92
Biosynthesis of phenylpropanoids	ko01061	5	14	26	92
Biosynthesis of secondary metabolites	ko01110	10	32	26	92
Anthocyanin biosynthesis	ko00942	3	8	26	92
Metabolic pathways	ko01100	6	16	26	92
Flavone and flavonol biosynthesis	ko00944	4	21	26	92
AMPK signaling pathway	ko04152	1	2	26	92
Isoflavonoid biosynthesis	ko00943	1	9	26	92
The compared transcriptome of HJ VS XN on KEGG metabolic pathway annotation
Pathway	ko_ID	Unique compound	compound	Uni_all	compound_all
Flavone and flavonol biosynthesis	ko00944	3	21	19	92
Isoflavonoid biosynthesis	ko00943	2	9	19	92
Biosynthesis of phenylpropanoids	ko01061	2	14	19	92
Biosynthesis of secondary metabolites	ko01110	5	32	19	92
Flavonoid biosynthesis	ko00941	9	36	19	92
Anthocyanin biosynthesis	ko00942	3	8	19	92
Metabolic pathways	ko01100	2	16	19	92
The compared transcriptome of HJ VS XX on KEGG metabolic pathway annotation
Pathway	ko_ID	Unique compound	compound	Uni_all	compound_all
Flavone and flavonol biosynthesis	ko00944	4	20	20	92
Anthocyanin biosynthesis	ko00942	6	8	20	92
Metabolic pathways	ko01100	5	16	20	92
Biosynthesis of secondary metabolites	ko01110	8	32	20	92
Flavonoid biosynthesis	ko00941	9	36	20	92
Biosynthesis of phenylpropanoids	ko01061	3	14	20	92
AMPK signaling pathway	ko04152	1	2	20	92
Isoflavonoid biosynthesis	ko00943	1	10	20	92
The compared transcriptome of XN VS XF on KEGG metabolic pathway annotation
Pathway	ko_ID	Unique compound	compound	Uni_all	compound_all
Flavone and flavonol biosynthesis	ko00944	2	21	27	91
Flavonoid biosynthesis	ko00941	14	35	27	91
Biosynthesis of phenylpropanoids	ko01061	1	13	27	91
Biosynthesis of secondary metabolites	ko01110	7	31	27	91
Anthocyanin biosynthesis	ko00942	3	7	27	91
Metabolic pathways	ko01100	1	15	27	91
Isoflavonoid biosynthesis	ko00943	1	9	27	91
The compared transcriptome of XN VS XX on KEGG metabolic pathway annotation
Pathway	ko_ID	Unique compound	compound	Uni_all	compound_all
Flavone and flavonol biosynthesis	ko00944	4	21	21	92
Anthocyanin biosynthesis	ko00942	5	7	21	92
Metabolic pathways	ko01100	1	15	21	92
Biosynthesis of secondary metabolites	ko01110	2	31	21	92
Flavonoid biosynthesis	ko00941	5	35	21	92
Isoflavonoid biosynthesis	ko00943	1	10	21	92
The compared transcriptome of XF VS XX on KEGG metabolic pathway annotation
Pathway	ko_ID	Unique compound	compound	Uni_all	compound_all
Flavone and flavonol biosynthesis	ko00944	2	21	16	92
Anthocyanin biosynthesis	ko00942	6	7	16	92
Flavonoid biosynthesis	ko00941	3	35	16	92
Biosynthesis of secondary metabolites	ko01110	1	31	16	92
Isoflavonoid biosynthesis	ko00943	1	10	16	92

 

Q9: L442-444, unclear. The up- and down-regulated DEGs for 5 compare combinations?

      Response: Thanks for the above suggestion. We apologize for the mistake. In the revised manuscript, based on your suggestion, we have changed “A total of 8,713, 17,085, 11,818, 17,652, 9,803, 17,687, 13,412, 15,772, 1,412, 16,058, 7,854, and 7,521 differential expression transcripts (DETs) were identified in XNFJ VS LDPF, XNFJ VS XNNC, XNFJ VS XNXY, XNNC VS XNXY, and XNXY VS LDPF compared combinations, respectively (Figure 4E).” to “A total of 8,713, 17,085, 11,818, 17,652, 9,803, 17,687, 13,412, 15,772, 1,412, 16,058, 7,854, and 7,521 differential expression transcripts (DETs) were identified in XF VS HJ, XF VS XN, XF VS XX, XN VS HJ, XN VS XX, and XX VS HJ compared combinations, respectively (Figure 4E).” in Line 459 to Line 461.

 

Q10: L537, The figure of correlation analysis could be presented in the manuscript. structural gene—metabolites, TFs—metabolites, structural gene—TFs.

      Response: Thanks for the above suggestion. We carefully check the differential expression of transcription factors between each group combined with transcriptome and metabolome. We have shown the relationship between the structural gene and metabolites in Figure S17 and Figure S18.

 

 

Figure S17: Correlation analyses of differentially expressed transcripts (DETs) involved in flavo-noids

 

 

 

Figure S18: Correlation analyses of differentially expressed transcripts (DETs) involved in anthocyanins

 

Q11: L558-561, List the index in the Result section (Same as No. 3)

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have changed “XNFJ had the highest total anthocyanins and the redness color of a* value, while XNXY had the lowest total anthocyanins and lightness. It might be the main reason for the transformation from white to pink to purple” to “Moreover, the content of anthocyanins is a vital factor influencing their flower coloring. XN had the highest total anthocyanins (XNNC, 264.96 mg/g of FW) and the redness color of a* value (a*, 4.52), while XX had the lowest total anthocyanins (23.03 mg/g of FW) and lightness (L*, 80.43). It might be the main reason for the transformation from white to pink to purple.” in Line 572 - Line 576.

 

Q12: 583-600, This part is redundant, just one or two sentences will be Okay. The authors should discuss the difference of metabolites, structural genes and TFs most, and then identify the key genes and TFs related to color modification in the petals of Loropetalum chinense and L. chinense var. rubrum.

      Response: Thanks for the above suggestion. We agree with your viewpoint of to discuss about the metabolites, structural genes, and then identify the key genes related to color modification in the petals of Loropetalum chinense and L. chinense var. rubrum, and we discussed it in paragraph two and paragraph four. We reported the full-length transcriptome of L. chinense var. rubrum for the first time, and we need to discuss its characteristic and compare with other plants.

 

Q13: The Methods section is too detailed and should be condensed.

      Response: Thanks for the above suggestion. We apologize for we can’t condense the methods section. The reviewer 1, reviewer 3 and reviewer 4 are all mentioned to supplement methods in more detailed. And the document is just one copy with loading.

 

Q14: The authors listed 34 tables and 18 figures in the supplementary materials. Some figures and tables should be combined, like Fig. -S14, Table S2-S7, S9-S14, S15-S20…

      Response: Thanks for the above suggestion. We agree with your viewpoint that we have provide many information on transcriptome, full-length transcriptome and metabolome. However, we think provide more detail information will be convenient to reads to look for what they want.

 

Q15: The order of four cultivars should be consistent.

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have changed all in our manuscript.

 

Q16: There are two colors in the petals of LDPF. The authors should explain the process of sample collection.

      Response: Thanks for the above suggestion. We have described the process of sample collection in our manuscript “The flowers (flowers in full bloom for one day), stems, leaves, and roots of HJ were har-vested, respectively, for Iso-Seq library construction. The flowers of XX, HJ, XF, and XN were also collected to total anthocyanin detection and sequencing library construction.” in Line 124 - Line 127. Huaye Jimu No.2 (HJ, S-SV-LC-060-2010) is a bud variant selected by our research group.

 

Once again, thank you very much for your comments and suggestions. A revised manuscript is attached. Should you have any questions, please contact us without any hesitation.

Sincerely yours,

Corresponding author

Yanlin Li

30^th April, 2023

Author Response File: Author Response.pdf

Reviewer 3 Report

This manuscript describes the transcriptomic and metabolomic analysis of flower color in the traditional ornamental plants, Loropetalum chinense. Authors attempted to elucidate flower color formation of four samples by investigating flavonoid components and exhaustive tanscriptomic profiling analysis in this plant species. The results are informative to understand the flower coloring mechanism in L. chinense. Therefore, this paper is worthy of publication in Agronomy.

I give some specific comments to improve the manuscript below.

Specific comments:

 

1.  Why don’t authors cite their own recent paper, Zhang et al. (BMC Plant Biol (2023) 23:133)? Different leaf color of Loropetalum chinense was examined in that paper and highly related to present manuscript. Please cite and discuss suitably.

  

2.

L88-89 

   In L. chinense var. rubrum, several genes involved in flavonoid biosynthesis have been identified. Are these corresponding to genes of present studies? Authors identified more genes, e.g. 20 CHS, 8 DFR (Fig 5B).  Regarding with this, I don’t understand the genes identified by transcriptome analyses in this study really exist in all used plants. Are these homologues (isotigs) or alleles? Did author check their presence in each plant sample? Please specify.

 

3.  Most data are derived from bioinformatic analysis in this paper. Did author check some of the sequences or expression levels by Sanger sequencing or RT-qPCR analysis? Is there genomic information available in this plant species or related species?

 

3.  Anthocyanin contents are most responsible for flower color. Authors present total anthocyanin contents (Fig. 1B) and each anthocyanin (Supplementary data). I recommend authors to summarize contents of each 15 anthocyanin among four samples as graph in Figure 1C. Color is partly explainable by the accumulated types of aglycones (cyanidin vs delphindin vs pelargonidin).

 

4.　Are there any genes involved in flavonoid transport? Some ABC, MATE, GSTs are reported responsible for anthocyanin accumulation in vacuoles. If possible, please add the genes to analysis.

 

 5.  Finally, how do authors consider which genes are keys in the different color?    Did authors find any mutations in the possible genes?

 

 

Minor points:

 

Authors defined each plant by four alphabets. It is somewhat difficult to follow.        I recommend authors to present them by two alphabets.  'Xiangnong Xiangyun', XX, 'Huaye Jimu 2', HJ; 'Xiangnong Fenjiao', XF; 'Xiangnong Nichang', XN.

 

 

L81   dihydro flavonols -> dihydroflavonols

L 83   3'-hydro flavonols  ->  3'-hydroxylase

L 84   dihydro flavonol   ->  dihydroflavonol

L 85   anthocyanin synthase -> anthocyanidin

L85    antho-cyanins   -> anthocyanidins and anthocyanins

L191, L381, L622   L. chinense    ->   italic

L220  　RAN　　　 ->　　RNA

L347  Is Apiin also flavone?  If so,   Flavonoid   -> flavone

L349   Anthocyanin (Cyanidin)  ->　 Anthocyanidin (Cyanidin)

L502, Fig. 5B     LcCYP73A -> C4H   

Fig. 5A   CYP75B1 -> F3'H   CYP75A  ->  F3'5'H   

         Flavone and flavonol biosynthesis  -> Flavone biosynthesis

         Please move Flavonol biosynthesis to FLS. 

I have no comments on Quality of English Language.

Author Response

Dear Reviewer,

 

Thank you for your detailed review of our manuscript entitled “Transcriptomic and metabolomic profiling provides insights into flavonoids biosynthesis and flower coloring in Loropetalum chinense and Loropetalum chinense var. rubrum” (2252556). The comments are of great help to improving the manuscript. We have studied the comments carefully and perform corresponding corrections in the revised manuscript. The point-by-point responses to the comments and suggestions are listed below.

 

This manuscript describes the transcriptomic and metabolomic analysis of flower color in the traditional ornamental plants, Loropetalum chinense. Authors attempted to elucidate flower color formation of four samples by investigating flavonoid components and exhaustive tanscriptomic profiling analysis in this plant species. The results are informative to understand the flower coloring mechanism in L. chinense. Therefore, this paper is worthy of publication in Agronomy. I give some specific comments to improve the manuscript below.

(1)  Why don’t authors cite their own recent paper, Zhang et al. (BMC Plant Biol (2023) 23:133)? Different leaf color of Loropetalum chinense was examined in that paper and highly related to present manuscript. Please cite and discuss suitably.

(2) L88-89

     In L. chinense var. rubrum, several genes involved in flavonoid biosynthesis have been identified. Are these corresponding to genes of present studies? Authors identified more genes, e.g. 20 CHS, 8 DFR (Fig 5B).  Regarding with this, I don’t understand the genes identified by transcriptome analyses in this study really exist in all used plants. Are these homologues (isotigs) or alleles? Did author check their presence in each plant sample? Please specify.

(3). Most data are derived from bioinformatic analysis in this paper. Did author check some of the sequences or expression levels by Sanger sequencing or RT-qPCR analysis? Is there genomic information available in this plant species or related species?

(4) Anthocyanin contents are most responsible for flower color. Authors present total anthocyanin contents (Fig. 1B) and each anthocyanin (Supplementary data). I recommend authors to summarize contents of each 15 anthocyanin among four samples as graph in Figure 1C. Color is partly explainable by the accumulated types of aglycones (cyanidin vs delphindin vs pelargonidin).

(5) Are there any genes involved in flavonoid transport? Some ABC, MATE, GSTs are reported responsible for anthocyanin accumulation in vacuoles. If possible, please add the genes to analysis.

(6) Finally, how do authors consider which genes are keys in the different color?    Did authors find any mutations in the possible genes?

(7) Authors defined each plant by four alphabets. It is somewhat difficult to follow.        I recommend authors to present them by two alphabets.  'Xiangnong Xiangyun', XX, 'Huaye Jimu 2', HJ; 'Xiangnong Fenjiao', XF; 'Xiangnong Nichang', XN.

(8) L81   dihydro flavonols -> dihydroflavonols.

(9) L83   3'-hydro flavonols  ->  3'-hydroxylase.

(10) L84   dihydro flavonol   ->  dihydroflavonol.

(11) L85   anthocyanin synthase -> anthocyanidin.

(12) L85    antho-cyanins   -> anthocyanidins and anthocyanins.

(13) L191, L381, L622   L. chinense    ->   italic

(14) L220  　RAN　　　 ->　　RNA

(15) L347  Is Apiin also flavone?  If so,   Flavonoid   -> flavone.

(16) L349   Anthocyanin (Cyanidin)  ->　 Anthocyanidin (Cyanidin).

(17) L502, Fig. 5B     LcCYP73A -> C4H.

(18) Fig. 5A   CYP75B1 -> F3'H   CYP75A  ->  F3'5'H  

         Flavone and flavonol biosynthesis  -> Flavone biosynthesis

         Please move Flavonol biosynthesis to FLS.

 

   Q1: Why don’t authors cite their own recent paper, Zhang et al. (BMC Plant Biol (2023) 23:133)? Different leaf color of Loropetalum chinense was examined in that paper and highly related to present manuscript. Please cite and discuss suitably.

Response: Thanks for the above suggestion and your attention to our group’s new progress in Loropetalum spp.. In the revised manuscript, based on your suggestion, we cited our newest result in our manuscript. We discussed it in a suitable location, such as Line 567- Line 568: “and the total anthocyanins of PL is much higher than ML and GL, and the lowest is GL”. And Line 570 -Line 571.

 

Q2: L88-89 In L. chinense var. rubrum, several genes involved in flavonoid biosynthesis have been identified. Are these corresponding to genes of present studies? Authors identified more genes, e.g. 20 CHS, 8 DFR (Fig 5B).  Regarding with this, I don’t understand the genes identified by transcriptome analyses in this study really exist in all used plants. Are these homologues (isotigs) or alleles? Did author check their presence in each plant sample? Please specify.

      Response: Thanks for the above suggestion. Firstly, all the genes by previously reported are partial CDS genes and can’t be searched from the NCBI or other public data. And their gene functions weren’t identified, so we can’t give you a positive answer. Secondly, we have finished the genome of Lorpetalum chinense var. rubrum. About 60 thousand genes have been annotated, and the results will be published for some time. There are many homologous genes or alleles that exist. Let us show the expression pattern of these genes (Table 1), we have check all the gene expression of selected samples.

Table 1 The FPKM value of identification of genes in Figure 5

Gene	XF1	XF2	XF3	XN1	XN2	XN3	XX1	XX2	XX3	HJ1	HJ2	HJ3
TCMO1	0.66	1.68	1.23	0	0	0.25	0	0	0	0	0	0.11
TCMO2	21.37	26.24	27.13	24.22	26.92	24.42	0.67	0	0	0	0	0.19
TCMO3	80.07	101.46	79.31	37.49	77.09	36.74	78.12	37.8	61.21	127.75	73.43	66.12
TCMO4	0.16	0.35	0.07	0.02	0.03	0.03	0.94	0.42	0.52	0.56	0.37	0.19
TCMO5	0	0	0	76.12	71.18	99.75	0	0	0	0	0	0
TCMO6	0	0	0	0.03	0	0.12	7.36	3.12	4.92	2.87	2.22	1.71
TCMO7	0.77	0.53	0.35	0.94	0.6	0.54	0.32	0.45	0.14	0	0.15	0.32
TCMO8	0.72	0.16	0.16	0.11	0.36	0.12	0	0	0	0	0	0
CHS1	4.56	10.05	5.84	33.54	43.95	58.62	22.97	7.73	13.14	0	0	0
CHS2	77.5	31.14	41.21	0	0	0	89.7	135.96	98.86	0.76	0.43	0.29
CHS3	347.95	259.4	375.33	451.23	503.8	512.72	633.22	373.64	479.24	599.72	672.16	846.28
CHS4	117.75	161.44	152.02	670.16	846.37	848.99	489.59	183.05	312.89	135.77	151.59	121.93
CHS5	92.48	134.69	144.4	0.08	0.04	0.09	45.31	8.47	28.77	0	0	7.92
CHS6	58.69	45.49	62.85	1.66	1.27	2.25	0.24	0.11	0.09	1.1	1.56	1.91
CHS7	5.73	3.32	7.78	14.78	14.69	13.29	10.4	0.71	7.85	1.99	1.54	1.07
CHS8	208.67	329.83	295.49	0.12	0.22	0.07	4.6	1.09	1.44	0.47	0.54	0
CHS9	3.13	7.47	5.09	0.1	0.1	0.22	0	0.3	0	0.06	0	0.15
CHS10	314.14	238.02	347.12	331.55	401.93	413.2	2.02	0.5	1.24	298.62	360.07	443.32
CHS11	2.69	1.78	1.27	0	0	0	2.05	5.98	3.03	2.6	1.39	0.98
CHS12	1.03	0.43	0.79	0	0	0	0.82	0.46	4.33	0	0	0
CHS13	0.54	0.7	0	0	0	0	1.01	2.42	2.57	0	0	0
CHS14	9.71	6.87	7.86	0.07	0	0.13	16.28	27.24	19.92	1.17	1.4	1.8
CHS15	14.84	11.44	16.46	1.5	0.8	1.29	0.77	0.29	0.16	1.24	0.82	0.94
CHS16	11.8	3.38	8.02	0	0	0	4.24	5.96	2.56	0	0	0
CHS17	1.84	0.39	0.87	2.6	1.15	1.95	5.29	2.7	2.39	2.11	2.85	2.73
CHS18	0.4	0.16	0.17	0.12	0.24	0.46	0	0	0	0	0.12	0.48
CHS19	5.45	8.08	4.2	0.22	0.13	0	11.56	9.68	12.45	21.58	15.57	7.47
CHS20	57.79	91.05	87.4	345.11	397.14	425.75	289.34	103.65	170.29	61.88	72.61	58.65
CFI1	42.84	54.95	49.16	68.05	75.7	78.18	53.81	33.99	37.63	34.79	35.8	41.87
CFI2	0.19	0	0	0.18	0.86	0.79	1.02	1.44	0.96	1.79	0.51	1.62
CFI3	0.36	0.41	0.43	0.29	0.27	0.2	0.85	0.66	0.71	0.3	0.43	0.43
FL3H1	4.73	5.72	3.37	0	0	0	0	0	0	1.27	0.79	0.45
FL3H2	1.1	1.57	0.4	26.93	30.17	27.38	26.83	13.69	16.19	20.69	18.79	15.79
FL3H3	2.02	2.79	1.4	0.3	0.28	0.12	0.62	1.09	0.72	1.9	0.59	0.25
FL3H4	0	0	0.09	3.57	3.27	2.94	3.66	0.96	2.21	3.2	2.56	3.5
FL3H5	2.08	0.7	1.17	0.12	0.15	0.19	0	0	0.04	0.67	0.2	0.13
FL3H6	8.79	22.54	17.34	105.17	128.46	100.54	78.18	26.04	49.64	17.48	22.41	21.96
FL3H7	18.05	35.35	21.29	117.09	150.29	138.26	43.55	16.89	22.46	9.5	12.32	7.38
FL3H8	3.19	4.77	4.01	1.44	1.9	0.93	2.99	1.58	0.74	4.57	1.67	0.96
FL3H9	3.24	3	2.12	0	0.01	0	0	0	0.08	0.84	0.32	0.32
FL3H10	25.42	34.78	29.22	168.98	204.67	176.01	82.13	25.34	42.34	50.54	42.33	45.96
FL3H11	1.27	1.09	2.09	0.11	0.39	0.11	1.89	0.85	0.67	0.18	0.31	0.63
FL3H12	1.12	1.8	0.72	8.73	8.56	8.46	7.68	2.21	4.01	5.14	5.62	5.24
FL3H13	0.55	0.54	0.27	0	0.08	0.06	0.08	0	0.14	0	0	0
FL3H14	0.41	0.59	0.67	0.76	0.69	1.11	0.25	0.64	0.49	0.37	0.19	0.03
FL3H15	4.1	6.18	5.09	21.62	25.07	23.28	1.08	0.54	0.73	0.23	1.32	1.43
FL3H16	0.84	0.86	1.59	1.95	0.67	1.38	9.36	3.29	4.78	1.03	0.85	0.78
FL3H17	0.42	0	0.07	2.15	0.65	1.83	0.49	0	0	0	0.03	0
FL3H18	0.07	0.04	0.43	0.62	0.36	0.38	1.78	0.7	0.23	0	0	0
FL3H19	1.36	1.64	0.69	2.02	2.92	2.63	2.29	0.59	0.86	2.21	0.66	0.66
DFRA1	13.77	22.5	17.33	41.69	46.76	47.19	37.27	8.44	15.42	9.32	6.39	4.21
DFRA2	24.82	45.36	42.47	94.97	108.03	118.29	106.52	24.9	59.47	37.13	45.14	30.98
DFRA3	0.94	1.58	2.52	6.78	7.61	6.43	3.67	1.2	1.72	2.73	0.65	0.69
DFRA4	66.18	146.26	130.33	425.87	478.84	535	348.48	67.29	165.76	18.39	26.47	12.05
DFRA5	39.87	87.91	72.99	243.34	302.94	319.69	207.84	39.12	95.96	8.45	13.83	7.01
DFRA6	6.52	10.46	8.47	0.41	0.27	0.59	1.11	0.29	0.32	0.6	0.1	0.19
DFRA7	0.52	0.4	0.6	0.33	0	0.07	0	0.06	0.06	0.15	0.07	0
DFRA8	0.71	1.28	1.27	1.87	2.12	1.67	2.52	0.51	0.62	0.14	0.09	0.17
LDOX1	13.18	27.1	17.04	115.73	121.87	109.6	67.69	9.28	24.34	12.56	13.56	5.75
LDOX2	0	0.54	0.55	0.42	0.53	0.45	0.63	0	0.29	0.11	0.08	0
LDOX3	19.4	47.72	25.18	190.2	224.74	203.36	132.83	21.28	46.54	3.41	3.89	5.77
LDOX4	0.46	0.74	0.59	1.02	0.86	1.06	0.74	0.25	0.55	0	0.37	0.06
F3PH1	64.75	83.43	77.49	274.84	320.88	333.59	133.7	64.48	92.35	63.58	72.65	67.69
F3PH2	6.54	9.09	6.95	1.11	0.99	1.55	3.31	2.14	1.17	2.84	0.24	0.55
F3PH3	48.59	50.93	55.83	9.82	11.33	36.09	8.08	1.47	5.38	7.03	6.58	2.5
F3PH4	63.88	68	66.03	93.41	111.95	85.65	241.1	123.64	166.16	79.2	91.22	99.4
F3PH5	0.8	0.73	0.65	0.95	0.98	0.93	0	0	0	0	0.15	0
F3PH6	0	0	0.04	2.17	2.51	3.65	3.08	1.48	2.79	1.21	0.65	0.95
F3PH7	0.64	0.69	0.38	1.75	1.28	2.7	3.43	1.51	1.49	0.72	1.1	0.82
F3PH8	0.26	0.54	0.26	2.37	1.99	2.36	1.31	0.45	0.23	0	0.7	0.16
F3PH9	0.17	0.13	0	0.29	0.21	0.5	0.39	0.11	0.1	0.04	0.03	0.06
F3PH10	13.56	12.35	16.11	0.33	0.22	0.19	0.34	0.08	0.33	0	0	0
F3PH11	2.69	1.65	1.36	1.8	1.9	1.98	6.34	3.36	4.06	1.45	2.06	1.93
UFOG1	21.07	12.46	16.38	5.24	1.68	2.66	0.26	0.42	0.12	9.01	4.23	3.88
UFOG2	43.04	29.58	46.91	23.51	32.18	28.85	85.84	80.09	82.82	42.45	52.68	66.7
UFOG3	1.13	1.92	1.31	1.21	0.94	3.38	1.4	0.51	0.85	0	0	0
UFOG4	1.87	1.09	1	1.08	0.56	0.41	1	0.25	0.95	0.79	0.33	0.47
UFOG5	1.03	1.31	0.18	3.67	5.58	2.15	0.81	0.94	3.72	0.18	0.78	0.19
UFOG6	0.68	1.18	0.63	1.27	2.21	3.68	3.53	1.64	1.98	0.1	0.1	0.17
UFOG7	27.16	30.44	31.47	2.19	2.1	1.51	0	0.09	0.08	2.64	2.17	3.56
UFOG8	2.28	4.39	1.45	0.4	0.82	0.82	1.98	1.1	1.46	1.86	1.18	1.07
UFOG9	2.81	2.67	4.89	0.66	0.9	0.74	0	0	0	0.07	0.08	0
UFOG10	0.37	0.06	0.08	0.04	0.04	0.07	1.26	1.53	1.47	0.3	0.31	0.6
UFOG11	0.49	0.26	0.11	0.75	0.14	0.43	0.78	0.79	1.23	20.26	12.34	8.71
UFOG12	19.29	19.96	21.39	4.55	5.41	4.75	0.07	0.06	0.13	2.71	1.95	3.44
3Mat	4.74	2.49	1.07	4.57	1.85	0.75	3.58	7.46	5.27	54.33	15.48	7.26
5Mat2	14.97	10.72	9.97	0.89	0.06	0.31	12.98	11	12.77	13.25	19.61	18.7
3AT1_1	1.24	0	0	1.65	0.83	0.52	0	0	0	5.09	1.58	1.4
3AT1_2	1.17	1.44	0.64	0.82	0.95	1.22	0.52	0	0	0.27	0.17	0
3AT1_3	1.38	1.95	0.55	1.58	1.3	0.93	1.93	2.33	1.86	8.32	3.05	1.9
3AT2	5.14	8.27	3.9	0.61	0.62	0.26	2.06	2.21	1.77	4.17	2.74	0.62
GT1_1	11.13	6	5.55	1.33	1.36	1.44	2.75	3.81	3.09	7.62	10.91	11.17
GT1_2	0.81	0.57	0.89	0.05	0	0	1.21	2.78	1.83	2.45	2.72	3.62

 

Q3: Most data are derived from bioinformatic analysis in this paper. Did author check some of the sequences or expression levels by Sanger sequencing or RT-qPCR analysis? Is there genomic information available in this plant species or related species?

      Response: Thanks for the above suggestion. We agree with your viewpoint, our manuscript is main on transcriptome and metabolome tools to provides insight into flavonoids biosynthesis and flower coloring in Loropetalum spp.. Up today, the transcriptome sequencing is a stable and reliable technology to do gene differential expression patter. It also accepted along and instead of fluorescence quantitative technique to by our other published articles (Xiong, X., Gou, J., Liao, Q., Li, Y., Zhou, Q., Bi, G., ... & Yan, J. (2021). The Taxus genome provides insights into paclitaxel biosynthesis. Nature Plants, 7(8), 1026-1036; Luo, Y., Deng, M., Zhang, X., Zhang, D., Cai, W., Long, Y., ... & Li, Y. (2023). Integrative Transcriptomic and Metabolomic Analysis Reveals the Molecular Mechanism of Red Maple (Acer rubrum L.) Leaf Coloring. Metabolites, 13(4), 464.).

      We have just finished the reference genome sequence of Lorpetalum chinensis var. rubrum assembly and annotation. And the results will be published later.

 

Q4: Anthocyanin contents are most responsible for flower color. Authors present total anthocyanin contents (Fig. 1B) and each anthocyanin (Supplementary data). I recommend authors to summarize contents of each 15 anthocyanin among four samples as graph in Figure 1C. Color is partly explainable by the accumulated types of aglycones (cyanidin vs delphindin vs pelargonidin).

      Response: Thanks for the above suggestion. We agree with your viewpoint if we detect the concentration of the 15 anthocyanin components among the four samples. However, for our metabolome data of flavonoids, we can’t summarize the 15-identifed anthocyanin among four samples. Firstly, we get data by using a widely targeted metabolome method by Wuhan Metware Biotechnology (Han Li, Zhen Yang, Qiwei Zeng, Shibo Wang, Yiwei Luo, Yan Huang, Youchao Xin, Ningjia He, Abnormal expression of bHLH3 disrupts a flavonoid homeostasis network, causing differences in pigment composition among mulberry fruits, Horticulture Research, Volume 7, 2020, 83, https://doi.org/10.1038/s41438-020-0302-8; Li, Y., Chen, Y., Zhou, L., You, S., Deng, H., Chen, Y., ... & Zhang, Y. (2020). MicroTom metabolic network: rewiring tomato metabolic regulatory network throughout the growth cycle. Molecular plant, 13(8), 1203-1218.). This protocol of qualitative and qualification of flavonoid is kind of semiquantitative, different flavonoid components summarized as index has no biological significance. Secondly, the protocol of total anthocyanin content detective is mainly index of all anthocyanidins content, it included of cyanidin, delphindin, pelargonidin, and other anthocyanidins. And we get the heatmap of 15-anthocyanins

content as can be seen from Figure S19.

Figure S19 The heatmap of 15 anthocyanins content among four samples

Notes: HJ: “Huaye Jimu 2hao”; XN: “Xingnong Nichang”; XF: “Xiangong Fenjiao”; XX: “Xingnong Xiangyun”

 

Q5: Are there any genes involved in flavonoid transport? Some ABC, MATE, GSTs are reported responsible for anthocyanin accumulation in vacuoles. If possible, please add the genes to analysis.

      Response: Thanks for the above suggestion. And we agree with you, the anthocyanins accumulation in vacuoles is key factor that affect the color of petals and leaves in plant. While we have checked our data, and we didn’t find the gene of ABC MATE and GSTs different expression among four samples. It maybe the biosynthesis of anthocyanin caused the concentration of anthocyanins in Loropetalum chinense var. rubrum and L. chinense.

 

Q6: Finally, how do authors consider which genes are keys in the different color?    Did authors find any mutations in the possible genes?

      Response: Thanks for the above suggestion. We have suggested the candidate gene in our manuscript in Line 635 – Line 638: “The genes of UFOG1, UFOG7, UFOG9, UFOG12, and 3AT1_2 were relatively higher ex-pressed in darker petals of samples. Furthermore, the UFOG2, UFOG10, and GT1_2 positively correlated with the delphinidin content.”. Frankly speaking, we don’t clone gene of each species, and this is huge work, but we find the significant expression among different species from Figure 6 in our manuscript and Table 1.

 

Q7: Authors defined each plant by four alphabets. It is somewhat difficult to follow.        I recommend authors to present them by two alphabets.  'Xiangnong Xiangyun', XX, 'Huaye Jimu 2', HJ; 'Xiangnong Fenjiao', XF; 'Xiangnong Nichang', XN.

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we redescribed all the names by two alphabets. Like  'Xiangnong Xiangyun', XX, 'Huaye Jimu 2', HJ; 'Xiangnong Fenjiao', XF; 'Xiangnong Nichang', XN.

 

Q8: L81   dihydro flavonols -> dihydroflavonols.

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have changed “dihydro flavonols” to “dihydroflavonols” in Line 82.

 

Q9: L83   3'-hydro flavonols  ->  3'-hydroxylase.

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have changed “3'-hydro flavonols” to “3'-hydroxylase” in Line 84.

 

Q10: L84   dihydro flavonol   ->  dihydroflavonol.

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have changed “dihydro flavonol” to “dihydroflavonol” in Line 82.

 

Q11: L85   anthocyanin synthase -> anthocyanidin.

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have changed “anthocyanin synthase” to “anthocyanidin” in Line 86.

 

Q12: L85    antho-cyanins   -> anthocyanidins and anthocyanins.

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have changed “anthocyanins” to “anthocyanidins and anthocyanins” in Line 86.

 

Q13: L191, L381, L622   L. chinense    ->   italic

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have changed all the L. chinense as italic in whole of our manuscript.

 

Q14: L220  　RAN　　　 ->　　RNA

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have changed the RNA to “RNA” in Line 233.

 

Q15: L347  Is Apiin also flavone?  If so,   Flavonoid   -> flavone.

      Response: Thanks for the above suggestion. We agree with your viewpoint. In the revised manuscript, based on your suggestion, we have changed the subclassification of “Apiin” from flavonoid to flavone Line 324, 356, and 384.

 

Q16: L349   Anthocyanin (Cyanidin)  ->　 Anthocyanidin (Cyanidin).

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have changed Anthocyanin (Cyanidin) to “three Anthocyanidin (Peonidin, Petunidin 3-O-glucoside, and Peonidin 3-sophoroside-5-glucoside)” in Line 360 – Line 361.

 

Q17: L502, Fig. 5B     LcCYP73A -> C4H.

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have changed the LcCYP73A to C4H in Figure 5B.

 

Q18: Fig. 5A   CYP75B1 -> F3'H   CYP75A  ->  F3'5'H  

         Flavone and flavonol biosynthesis  -> Flavone biosynthesis

         Please move Flavonol biosynthesis to FLS.

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we redraw the Figure 5, and changed the pathway according to your advice.

 

 

Figure 5. Simplified representation of flavonoid metabolism and heat map produced by significant different gene related to flavonoid synthetic of L. chinense var. rubrum. (A). Simplified representation of flavonoid metabolism. Red rectangular boxes represented significant changed metabolism according to KEGG enrichment analysis. (B). Heat map produced by significant different gene related to flavonoid synthetic. And the gray rectangular boxes represented the enzyme coded by related genes. The orange square represented upregulated, and purple square represented downregulated. White square represented the gene related to flavonoid synthetic that hadn't significant changed.

 

Once again, thank you very much for your comments and suggestions. A revised manuscript is attached. Should you have any questions, please contact us without any hesitation.

Sincerely yours,

Corresponding author

Yanlin Li

27^th April, 2023

Author Response File: Author Response.pdf

Reviewer 4 Report

In this work the anthocyanins and flavonoids analysis and transcriptomic expression related to the colour differences observed in Loropetalum chinense and Loropetalum chinense var. rubrum have been carried out. Although the work is of undoubted interest, to improve the quality, a general English correction is needed to make the reading more fluent and remove minor errors. Below is a series of revisions/questions for authors:

L30-31: rewriting the sentence

-Transcriptomic analysis, not taking into account the different possible fates of mRNA, remains a predictive and potential analysis, would it not have been more appropriate to check the actual correspondence between proteome and metabolome?

L48: etc.

L58: add reference

L54-55: rewrite sentence

L 76 77: "conserved among the seeds" is not a correct way of expressing the concept. Rewriting the sentence in a scientifically sound manner

L 145: Centrifugation at room temperature?

L 192: poorly understandable, rewriting the sentence

L 194: How long did you store the RNA at -20°C before using it?

L 207: sequencguh?

L 285: this is more an information to be given in M&M rather than in discussion

 A general English correction is needed to make the reading more fluent and remove minor errors

Author Response

Dear Reviewer,

 

Thank you for your detailed review of our manuscript entitled “Transcriptomic and metabolomic profiling provides insights into flavonoids biosynthesis and flower coloring in Loropetalum chinense and Loropetalum chinense var. rubrum” (2252556). The comments are of great help to improving the manuscript. We have studied the comments carefully and perform corresponding corrections in the revised manuscript. The point-by-point responses to the comments and suggestions are listed below.

 

In this work the anthocyanins and flavonoids analysis and transcriptomic expression related to the colour differences observed in Loropetalum chinense and Loropetalum chinense var. rubrum have been carried out. Although the work is of undoubted interest, to improve the quality, a general English correction is needed to make the reading more fluent and remove minor errors. Below is a series of revisions/questions for authors:

(1) L30-31: rewriting the sentence

-Transcriptomic analysis, not taking into account the different possible fates of mRNA, remains a predictive and potential analysis, would it not have been more appropriate to check the actual correspondence between proteome and metabolome?

(2) L48: etc.

(3) L58: add reference

(4) L54-55: rewrite sentence

(5) L 76 77: "conserved among the seeds" is not a correct way of expressing the concept. Rewriting the sentence in a scientifically sound manner

(6) L 145: Centrifugation at room temperature?

(7) L 192: poorly understandable, rewriting the sentence

(8) L 194: How long did you store the RNA at -20°C before using it?

(9) L 207: sequencguh?

(10) L 285: this is more an information to be given in M&M rather than in discussion

 

   Q1: L30-31: rewriting the sentence -Transcriptomic analysis, not taking into account the different possible fates of mRNA, remains a predictive and potential analysis, would it not have been more appropriate to check the actual correspondence between proteome and metabolome?

Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have changed “Moreover, a total of 207 flavonoid components were identified. Of these, 12 flavonoid components were considered significantly different expression compounds among the four samples.” to “Moreover, a total of 207 flavonoid components were identified. Of these, 13 flavonoid compounds were considered significantly different expression compounds highly consistent with color information in the four samples.” In Line 30 – Line 32.

 

Q2: L48: etc.

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have “et al.” to “etc.” in Line 48

 

Q3: L58: add reference

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have added the references, and described as below: “while litter is known about the flavonoid biosynthesis in petals of L. chinense var. rubrum [5-7].” in Line 59.

 

Q4: L54-55: rewrite sentence

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have changed “The different distribution of anthocyanins in the petals of sponge tissue cells was considered the main reason for different petal coloring in L. chinense var. rubrum [5]” to “The different distribution of anthocyanins in the petals of parenchymal cells led to the petal coloring differences in L. chinense var. rubrum [5]” in Line 55 – Line 56.

 

Q5: L 76 77: "conserved among the seeds" is not a correct way of expressing the concept. Rewriting the sentence in a scientifically sound manner

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have changed “The flavonoid biosynthetic pathway is well understood and conserved among seeds in plants [18].” to “The flavonoid biosynthetic pathway is well understood in plants [19].” Line 77.

 

Q6: L 145: Centrifugation at room temperature?

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have added the centrifugation temperature and described as below: “The supernatant was transferred into a clean tube after centrifugation at 1000×g at 4 °C for 15 min.” in Line 151 – Line 152.

 

Q7: L 192: poorly understandable, rewriting the sentence

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have changed “The other was four flower cultivars (HJ, XNNY, XF, and XN) with twelve libraries sub-jected to 2×150 paired-end RNA-seq using ILLUMINA novaseq 6000.” to “Total RNA from the four samples (HJ, XNNY, XF, and XN) with three times replication were extracted, respectively. Twelve libraries were subjected to 2×150 paired-end RNA-seq using ILLUMINA novaseq 6000.” in Line 205 – Line 206.

 

Q8: L 194: How long did you store the RNA at -20°C before using it?

      Response: Within one week. We have changed it to “All the qualified RNA samples were stored at -20℃ and used for SMRT sequencing and RNA-seq within one week.” in Line 207 – Line 208.

 

Q9: L 207: sequencguh?

      Response: Thanks for the above suggestion. We apologize for the mistake, and we have changed it “sequencguh reads” to “The circular consensus sequence was generated from subread BAM files and then classified into the non-full length and full-length fasta files by using pbclassify.py with ignore polyA false and min Seq Length 200. Next, the isoform-level clustering (ICE) and final arrow polishing were fed into the clustering step following the parameter configuration of hq_quiver_min_accuracy 0.99, bin_by_primer false, bin_size_kb 1, qv_trim_5p 100, qv_trim_3q 30.” in Line 220 – Line 224.

 

Q10: L 285: this is more an information to be given in M&M rather than in discussion

      Response: Thanks for the above suggestion. In the revised manuscript, based on your suggestion, we have changed the described as below: “ Additionally, a surprising degree of anthocyanins accumulation was observed in the four Loropetalum cultivars, especially in XN and XF.” in Line  307 – Line 309 and “These results also showed that L. chinense var. rubrum was a critical resource of anthocyanins compared to Asparagus officinalis (22.04 mg/g of FW) [52], Vaccinium corymbosum (about 1.8 mg/g of FW) [78], Lycoris longituba (1.25 mg/g of FW) [79] and other horticultural plants.” in Line 576 – Line 579.

 

Once again, thank you very much for your comments and suggestions. A revised manuscript is attached. Should you have any questions, please contact us without any hesitation.

Sincerely yours,

Corresponding author

Yanlin Li

30^th April, 2023

Author Response File: Author Response.pdf